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ASBStandard036,FirstEdition2017
StandardPracticesforMethodValidationinForensicToxicology
ASBStandard036,1stEd.‐2017
StandardPracticesforMethodValidationinForensicToxicology
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ASBStandard036,1stEd.‐2017
Foreword
Validationistheprocessofperformingasetofexperimentstoestablishobjectiveevidencethatamethodisfitforpurpose,andtoidentifythemethod'slimitationsundernormaloperatingconditions.Thisstandardwasdevelopedtoprovideguidanceonminimumrequirementsforvalidatinganalyticalmethodsinforensictoxicologylaboratories.
Thisdocumentwasrevised,preparedandfinalizedasastandardbytheToxicologyConsensusBodyoftheAAFSASB.ThedocumentwasoriginallydraftedbytheScientificWorkingGrouponForensicToxicology(SWGTOX).WhenSWGTOXdisbandedin2014,itpassedownershipofallofitsdocumentstotheToxicologySubcommitteeoftheOrganizationofScientificAreaCommittees(OSAC)whointurnupdatedandapprovedthedraftdocument.Allhyperlinksandwebaddressesshowninthisdocumentarecurrentasofthepublicationdateofthisstandard.
Keywords:Validation,ForensicToxicology
Abstract:Thisstandardwasdevelopedtoprovideguidanceonminimumrequirementsforvalidatingmethodsinforensictoxicologylaboratories.Propermethodvalidationensuresthatalaboratoryestablishesobjectiveevidencethatamethodisfitforpurposeandthethemethod’slimitationsundernormaloperatingconditionsareunderstood.
ASBStandard036,1stEd.‐2017
Acknowledgements
BaseDraftDevelopedbytheOrganizationofScientificAreaCommittees(OSAC)ToxicologySubcommittee:
Editor: MarcA.LeBeau,PhDF‐ABFT;FederalBureauofInvestigation;LaboratoryDivision;Quantico,Virginia
WorkingGroup: NicholeBynum,MS;RTIInternational;ResearchTrianglePark,NorthCarolina
ConnieM.Borror,PhD;ArizonaStateUniversity;NewCollegeofInterdisciplinaryArtsandSciences;Phoenix,Arizona
SimonElliott,PhD;ROARForensics;Worcestershire,UnitedKingdom
MarilynA.Huestis,PhD;HuestisandSmithToxicology,SevernaPark,Maryland
MatthewP.Juhascik,PhDF‐ABFT;MontgomeryCountyCoroner’sOffice;Dayton,Ohio
JamesC.Kraner,PhD,F‐ABFT;OfficeoftheChiefMedicalExaminer;Charleston,WestVirginia
LoralieJ.Langman,PhD,F‐ABFT,DABCC;MayoClinic;Rochester,Minnesota
JenniferLimoges,MS,DABC;NewYorkStatePolice‐Forensics;Albany,NewYork
ChristineMoore,PhD,DABCC;ImmunalysisCorporation;Pomona,California
StephenL.Morgan,PhD;UniversityofSouthCarolina;DepartmentofChemistryandBiochemistry;Columbia,SouthCarolina
RobertJ.Osiewicz,PhDF‐ABFT;ErieCountyMedicalExaminer’sOffice(Retired);Buffalo,NewYork
JuliaPearson,PhDF‐ABFT;HillsboroughCountyMedicalExaminer’sOffice;Tampa,Florida
FrankT.Peters,PhD;InstitutfuerRechtsmedizin;UniversitaetsklinkumJena;JenaGermany
SumandeepRana,PhD;RedwoodToxicologyLaboratory;SantaRosa,California
MatthewSlawson,PhD;UtahDepartmentofHealth;Taylorsville,Utah
ElizabethSpratt,MSF‐ABFT;WestchesterCountyDepartmentofLaboratories;Valhalla,NewYork
ScientificWorkingGrouponForensicToxicology(SWGTOX)
ToxicologySubcommitteeoftheOrganizationofScientificAreaCommittees(OSAC)
FinalStandardPreparedandFinalizedbytheASBToxicologyConsensusBody:
Members: WendyAdams,Ph.D.,F‐ABFT;NMSLabs;WillowGrove,Pennsylvania
SabraBotch‐Jones,M.S.M.A.D‐ABFT‐FT;BostonUniversitySchoolofMedicine,BiomedicalForensicSciences;Boston,Massachusetts
ASBStandard036,1stEd.‐2017
RandalClouette,M.S.F.S.,D‐ABFT‐FD;QuestDiagnostics,Inc;Lenexa,Kansas
FionaCouper,Ph.D.,WashingtonStatePatrol;Seattle,Washington
KennethFerslew,Ph.D.,F‐ABFT;EastTennesseeStateUniversity;JohnsonCity,Tennessee
DeanFritch,Ph.D.,F‐ABFT;OraSureTechnologies,Inc;Bethlehem,Pennsylvania
ShannonGeorge,B.S.;IllinoisStatePolice;Springfield,Illinois
MicheleGlinn,Ph.D.,F‐ABFT;EssentialTestingLLC/Avertest;Collinsville,Illinois
JamesHutchison,Jr.,M.S.,D‐ABFT‐FT;MontanaForensicToxicologyServices,LLC;Lolo,Montana
MatthewJuhascik,Ph.D.,F‐ABFT;MontgomeryCountyCoroner’sOffice;Dayton,Ohio
PhilipKemp,Ph.D.,F‐ABFT;CivilAerospaceMedicalInstitute(FAA);OklahomaCity,Oklahoma
MelissaKennedy,M.S.,D‐ABFT‐FA;ANSI‐ASQNationalAccreditationBoard;Garner,NorthCarolina
MarcLeBeau,Ph.D.,F‐ABFT;FederalBureauofInvestigation;Quantico,Virginia
AdamNegrusz,Ph.D.,F‐ABFT;UnitedStatesDrugTestingLaboratories,Inc.;DesPlaines,Illinois
DouglasRohde,M.S.;LakeCountyCrimeLaboratory;Painesville,Ohio
RobertSears,M.S.,F‐ABFT;SouthCarolinaLawEnforcementDivision;Columbia,SouthCarolina
MichaelStypa,M.S.,D‐ABFT‐FT;LasVegasMetropolitanPoliceDepartment;LasVegas,Nevada
TedVosk,J.D.;Kirkland,Washington
FrankWallace,B.A.;AmeritoxLLC;HighPoint,NorthCarolina
DarcieWallace‐Duckworth,Ph.D.;AegisSciencesCorporation;Nashville,Tennessee
JeffWalterscheid,Ph.D.,F‐ABFT;ArmedForcesMedicalExaminerSystem;DoverAFB,Delaware
RuthWinecker,Ph.D.,F‐ABFT;OfficeoftheChiefMedicalExaminer;Raleigh,NorthCarolina
DustinTateYeatman,M.S.F‐ABFT,F‐ABC;PalmBeachCountySheriff’sOfficeCrimeLaboratory;WestPalmBeach,Florida
ASBStandard036,1stEd.‐2017
TableofContents 1. Scope.................................................................................................................................................................................1
2. NormativeReference..................................................................................................................................................1
3. TermsandDefinitions...............................................................................................................................................1
4. WhentoValidateMethods.......................................................................................................................................3
5. MethodDevelopmentandOptimization............................................................................................................3
6. EstablishingaValidationPlan................................................................................................................................4
7. RequiredValidationParametersBasedonScopeoftheMethod............................................................4
8. SpecificRequirementsforConductingMethodValidationExperiments.............................................5
8.1. BiasandPrecision..............................................................................................................................................6
8.2. CalibrationModel...............................................................................................................................................8
8.3. Carryover.............................................................................................................................................................10
8.4. InterferenceStudies........................................................................................................................................10
8.5. IonizationSuppression/Enhancement...................................................................................................11
8.6. LimitofDetection.............................................................................................................................................12
8.7. LowerLimitofQuantitation........................................................................................................................14
9. AdditionalValidationParameters.......................................................................................................................15
9.1 General..................................................................................................................................................................16
9.2 DilutionIntegrityStability............................................................................................................................16
9.3 Stability................................................................................................................................................................16
10. RequiredRevalidationofPreviouslyValidatedMethods.........................................................................17
11. DocumentationRequirementsforMethodValidation...............................................................................17
12. EfficiencywithValidation.......................................................................................................................................18
ANNEXA:QuantitationofDrugXinBloodValidationExample......................................................................19
ANNEXB:ImmunoassayScreenofBenzodiazepinesinUrineValidationExample................................32
ANNEXC:ExampleFlowchartofMethodValidationExperiments................................................................35
ANNEXD:TableofExampleExperimentsforValidationofQualitativeConfirmation/IdentificationMethods...................................................................................................................................................................................36
ANNEXE:TableofExampleExperimentsforValidationofQuantitativeMethods.................................37
ANNEXF:Bibliography.....................................................................................................................................................38
ASBStandard036,1stEd.‐2017
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StandardPracticesforMethodValidationinForensicToxicology
1. Scope
Thisdocumentdelineatesminimumstandardsofpracticeforvalidatinganalyticalmethodsinthefieldofforensictoxicology.Thefundamentalreasonforperformingmethodvalidationistoensureconfidenceandreliabilityinforensictoxicologicaltestresultsbydemonstratingthemethodisfitforitsintendeduse.
2. NormativeReferences
Therearenonormativereferences.AnnexG,Bibliography,containsinformativereferences.
3. TermsandDefinitions
Forpurposesofthisdocument,thefollowingdefinitionsapply.
3.1. biasAnestimateofsystematicmeasurementerror,calculatedasthedifferencebetweenthemeanofseveralmeasurementsunderidenticalconditions,toaknown“true”value.Itisoftenreportedasapercentdifference.
3.2. biologicalfluidsAnyliquidbiologicalspecimenthatistypicallypipettedforanalysis(e.g.,blood,urine,bile,serum,vitreoushumor,oralfluid).
3.3. blankmatrixsampleAbiologicalfluidortissue(orsyntheticsubstitute)withouttargetanalyteorinternalstandard.
3.4. calibrationmodelThemathematicalmodelthatdemonstratestherelationshipbetweentheconcentrationofanalyteandthecorrespondinginstrumentresponse.
3.5. carryoverTheappearanceofunintendedanalytesignalinsamplesaftertheanalysisofapositivesample.
3.6. decisionpointAnadministrativelydefinedcutofforconcentrationthatisatorabovethemethod’slimitofdetectionorlimitofquantitationandisusedtodiscriminatebetweenpositiveandnegativeresults.
3.7. fortifiedmatrixsampleAblankmatrixsamplespikedwithtargetanalyteand/orinternalstandardusingreferencematerials.
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3.8. interferencesNon‐targetedanalytes(i.e.,matrixcomponents,otherdrugsandmetabolites,internalstandard,impurities)whichmayimpacttheabilitytodetect,identify,orquantitateatargetedanalyte.
3.9. ionizationsuppression/enhancementDirectorindirectalterationorinterferenceintheinstrumentresponseduetothepresenceofco‐elutingcompounds.
3.10. limitofdetectionAnestimateofthelowestconcentrationofananalyteinasamplethatcanbereliablydifferentiatedfromblankmatrixandidentifiedbytheanalyticalmethod.
3.11. lowerlimitofquantitationAnestimateofthelowestconcentrationofananalyteinasamplethatcanbereliablymeasuredwithacceptablebiasandprecision.
3.12. precisionThemeasureoftheclosenessofagreementbetweenaseriesofmeasurementsobtainedfrommultiplesamplingsofthesamehomogenoussample.Itisexpressednumericallyasimprecision.
3.13. referencematerialMaterial,sufficientlyhomogenousandstablewithreferencetospecifiedproperties,whichhavebeenestablishedtobefitforitsintendeduseinameasurementorinexaminationofnominalproperties.
3.14. stabilityAnanalyte’sresistancetochemicalchangeinamatrixunderspecificconditionsforgiventimeintervals.
3.15. tissuesAnysolidbiologicalspecimenthatisgenerallyweighedforanalysis(e.g.,brain,liver,muscle,hair,meconium).
3.16. workingrangeTherangeofconcentrationthatcanbeadequatelydeterminedbyaninstrument,wheretheinstrumentprovidesausefulsignalthatcanberelatedtotheconcentrationoftheanalyte.
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4. WhentoValidateMethods
Methodsshallbevalidatedtoverifyamethod'sperformanceparametersarefitforuseforaparticularanalysis.Commonexamplesinclude:
a) newanalyticalmethod;
b) modificationsofanestablishedanalyticalmethodtoimproveperformanceorextenditsusebeyondthatforwhichitwasoriginallyvalidated(e.g.,additionofnewcompoundstothemethod’sscope);
c) todemonstrateequivalencebetweenanestablishedmethod/instrumentandanewmethod/instrument;
d) existinganalyticalmethodsthatdonotcurrentlymeettherequirementsofthisdocument.
Theparameterstobeevaluatedforvalidationofmethodswilldependuponthecircumstancesinwhichthemethodistobeused.Likewise,itisrecognizedthataftervalidationhasoccurred,methodsmayberevised.Theextentandfrequencyofrevalidationofpreviouslyvalidatedmethodswilldependuponthenatureoftheintendedchangesorlaboratorypolicy.SeeSection10forfurtherguidanceonrevalidationofpreviouslyvalidatedmethods.
Laboratoriesusingmethodsthatwerevalidatedpriortothepromulgationoftheseminimumstandardsshalldemonstrateanddocumentthatthesemethodsarefitforpurposeunderthesestandards.Thesemethodswilllikelyhavesufficienthistoricalcalibrationandcontroldata,aswellaspreviouslyanalyzedcaseworksampleresults,thatcanbeusedtoaddressanumberoftherequiredvalidationparameters.Whensufficientdataareabsenttofulfilltheseminimumstandards,appropriatestudiesshallbeconductedtoensurecompliancewiththisdocument.
5. MethodDevelopmentandOptimization
5.1. General
Forpurposesofthisdocument,methoddevelopmentwillbeconsideredintwophases:1)instrumentalanddataacquisition/processingparametersand2)samplepreparation.Itisessentialthatvalidationisconductedwiththesameanalyticalconditionsandtechniquesasthefinalmethod.
Theprinciplesofgoodlaboratorypracticeandrecordkeepingshallbeappliedtotheconceptsofthisdocument.Thisincludesdocumentationofparametersthatwereevaluatedduringmethoddevelopment,yetdidnotprovideacceptableresults.
5.2. DevelopmentandOptimizationofInstrumentalandDataProcessingParameters
Instrumentalanddataprocessingparametersaredefinedandoptimizedthroughanalysisofreferencematerialsoftheanalyte(s)ofinteresttoachievetherequiredperformanceoftheinstrument.
5.3. DevelopmentandOptimizationofSamplePreparationTechniques
Thesamplepreparationtechniqueshallbeevaluatedandoptimizedusingreferencematerialsoftheanalyte(s)ofinterest.Theprimarygoalistodemonstratethatthesamplepreparationsteps
ASBStandard036,1stEd.‐2017
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allowforadequateextraction,detection,identification,and/orquantitationoftheanalyte(s).Samplepreparationshallbeevaluatedwithfortifiedmatrixsamples.
6. EstablishingaValidationPlan
Thelaboratoryisresponsibleforensuringitsmethodsareadequatelyvalidated.Avalidationplanshallbeinplacepriortostartinganyvalidationexperiments.Thevalidationplanisseparatefromalaboratory’sstandardoperatingprocedure(SOP)formethodvalidation.Theplanshallincludetheinstrumentalmethod(s)andsamplepreparationtechnique(s)tobeusedforaspecificmethod.Further,itshalldocumentthevalidationrequirementsofthemethod,aswellasthelimitsofthemethodthatwillallowittobefitforuse.Thevalidationplanprovidesdirectionfortheexperimentsthatwillbeperformedandacceptancecriteriaforeachparameter.AnnexBandAnnexCprovideexamplesofvalidationplans.
7. RequiredValidationParametersBasedonScopeoftheMethod
7.1. Thescopeofforensictoxicologymethodsistypicallycategorizedasscreeningmethods,qualitativeconfirmation/identificationmethods,orquantitativemethods.Assuchthefollowingvalidationparametersshallbeevaluated.
7.2. Screening(Immunoassay‐based):
a) limitofdetection;
b) precision(atthedecisionpoint);
c) dilutionintegrity(ifapplicable);
d) stability(ifapplicable).
7.3. Screening(allothers):
a) interferencestudies;
b) limitofdetection;
c) ionizationsuppression/enhancement(forapplicabletechniques,suchasLC/MS);
d) dilutionintegrity(ifapplicable);
e) stability(ifapplicable).
7.4. Qualitativeconfirmation/identification:
a) carryover;
b) interferencestudies;
c) ionizationsuppression/enhancement[forapplicabletechniques,suchasliquidchromatography/massspectrometry(LC/MS)];
ASBStandard036,1stEd.‐2017
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d) limitofdetection;
e) dilutionintegrity(ifapplicable);
f) stability(ifapplicable).
7.5. Quantitativeanalysis:
a) bias;
b) calibrationmodel;
c) carryover;
d) interferencestudies;
e) ionizationsuppression/enhancement(forapplicabletechniques,suchasLC/MS);
f) limitofdetection;
g) limitofquantitation;
h) precision;
i) dilutionintegrity(ifapplicable);
j) stability(ifapplicable).
8. SpecificRequirementsforConductingMethodValidationExperiments
8.1. General
Allvalidationexperimentsshallbeconductedusingfortifiedsamplesofthematrixforwhichthemethodisintended,unlessotherwisenoted.Insomeinstances(e.g.,immunoassayscreens),itmaybemoreappropriatetoanalyzepreviouslycharacterizedhumansamplesinsteadoffortifiedsamplesforselectedmethodvalidationstudies.
Validationstudiesshallbeconductedinamannersimilartocasework.Thismayincludeconductingvalidationstudiesondifferentdays,bydifferentanalysts,onallidenticalinstrumentstobeutilizedfortheassay,andensuringthattheinstrumentsmeetthesamedailyperformancerequirementsasforcasework.
Wheneverpossible,fortifiedmatrixsamplesshallbepreparedfromreferencematerialsthatarefromadifferentsource(e.g.,supplierorlotnumber)thanusedtopreparecalibrationsamples.Ininstanceswherethesamesourceshallbeutilized,separateweighingsorsolutionsshallbeusedtopreparethesesamples.
Thefollowingrequirementsaretheminimumforassessingthelistedvalidationparametersinforensictoxicologymethods.Theyarelistedalphabeticallyandnotnecessarilyinproceduralorder.SomeofthevalidationexperimentsaredemonstratedinAnnexBandAnnexC.Section11providesguidanceonhowtoefficientlyperformvalidationexperiments.
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8.2. BiasandPrecision1
8.2.1. Bias
Biasstudiesshallbecarriedoutforallquantitativemethods.Thesecanbeconductedconcurrentlywithprecisionstudies.Biasshallbemeasuredinpooledfortifiedmatrixsamplesusingaminimumofthreeseparatesamplesperconcentrationatthreedifferentconcentrationpools(low,mediumandhigh2)overfivedifferentruns.Thebiasshallbecalculatedforeachconcentrationusingthefollowingformula:
%
Themaximumacceptablebiasis±20%ateachconcentration.Forsomeanalyseswherelessbiasisrequired(e.g.,ethanol),abiasof±10%orbetterisexpected.Itisrecommendedthatthesamedatausedinbiasstudiesalsobeusedforprecisioncalculations.
8.2.2. Precision
8.2.2.1. General
Precisionstudiesshallbecarriedoutforallquantitativemethods,aswellasatthedecisionpointforimmunoassays.Thesestudiescanbecarriedoutconcurrentlywithbiasstudies,ifrequiredinthevalidationplan.
Precisionisexpressedasthecoefficientofvariation(%CV).Themeanandstandarddeviation(s)oftheresponseiscalculatedforeachconcentrationtodeterminethe%CV.
%
8.2.2.2. PrecisionofImmunoassaysatDecisionPoint
Forimmunoassaysthatcross‐reactwithabroadclassofcompounds(e.g.,benzodiazepines,opiates,amphetamines),ifalaboratorydeclarestotheircustomersthattheyareabletodetectanalyteswithlowcross‐reactivity(lessthanorequaltothetargetanalyte)usingtheimmunoassay,itisessentialtoverifytheirabilitytodetectthesecompounds.Forexample,abenzodiazepineimmunoassaytargetedforoxazepamtypicallyhaslowcross‐reactivitiestootherbenzodiazepinessuchaslorazepam.Ifthelaboratoryusesthisimmunoassaykittoscreenforlorazepam,theyarerequiredtoevaluatetheassay’sabilitytoreliablydetectlorazepam,inadditiontooxazepam.Incontrast,ifalprazolamhasgreatercross‐reactivitythanoxazepam,thereisnorequirementtoevaluatetheabilitytodetectalprazolamprovidedthatthedecisionconcentrationforalprazolamisnotlowerthanthedecisionconcentrationforoxazepam.Thisevaluationmayrequireanadjustment
1Anaccuratemeasurementisonewithacceptablebiasandprecision.2Forpurposesofthisdocument,lowconcentrationsshallbenomorethanapproximately3timesthelowestendoftheworkingrangeofthemethodandhighconcentrationsshallbewithinapproximately80%(ormore)ofthehighestendoftheworkingrangeofthemethod,unlessotherwisenoted.Mediumconcentrationsshallbenearthemidpointofthelowandhighconcentrations.
ASBStandard036,1stEd.‐2017
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orreevaluationofthedecisionpointorthetargetcompounddependingontheneedsandmissionofthelaboratory.
Ataminimum,precisionatthedecisionpointshallbeassessedusingthreeseparatesamplesfromthreedifferentconcentrationpoolsoverfivedifferentruns:
a) generallynomorethan50%belowdecisionpoint,
b) atdecisionpoint,and
c) generallynomorethan50%forurineand100%forallothermatricesabovedecisionpoint.
Immunoassaysarematrixdependentandtheconcentrationrangearoundthedecisionpointmaybewiderformorecomplexmatricesincomparisontourine.
EnzymeLinkedImmunosorbentAssays(ELISA):Thedifferencebetweentheabsorbanceofanegativesample(Bo)andtheabsorbanceofaspecimen(B)shouldbemeasuredasapercentage:[B/Bo]x100andnotanabsolutevalue.
Liquidreagentassays(e.g.enzymemultipliedimmunoassaytechnique[EMIT],clonedenzymedonorimmunoassay[CEDIA®3],etc.):Theabsorbancevaluecanbeuseddirectly.
a) Runeachconcentration3timesinfiveseparateruns.
b) Calculatethegrandmean(n=15)andrelatedgrandstandarddeviationateachconcentration.
c) The%CVshallnotexceed20%ateachconcentrationusingall15sampleresultsperconcentration.
d) Thegrandmeanplusorminustwostandarddeviations(stddev)foreachconcentrationshallnotoverlapforthedecisionpointtobevalid.
ItshouldbenotedthatthedataobtainedfromthesestudiesarealsousedtoestimatetheLODforimmunoassays.
8.2.2.3. PrecisionofQuantitativeProcedures
8.2.2.3.1. General
Forquantitativeprocedures,twodifferenttypesofprecisionstudiesshallbeassessedduringmethodvalidation:within‐runprecisionandbetween‐runprecision.Ataminimum,precisionshallbeassessedusingthreedifferentsamplesperconcentrationatthreedifferentconcentrationpools(low,mediumandhigh)overfivedifferentruns.Thedifferentrunsusedtoevaluateprecisionmaybeperformedwithinthesameday,providedadifferentcalibrationcurveisusedforeachrun.
The%CVshallnotexceed20%ateachconcentration.Itisnotedthatcertainanalyticalmethods(e.g.,bloodalcoholanalysis)mayrequireamuchlowercoefficientofvariation(≤10%CV).
3Thistermisusedasanexampleonly,anddoesnotconstituteanendorsementofthisproductbytheAAFSStandardsBoard.
ASBStandard036,1stEd.‐2017
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8.2.2.3.2. Within‐RunPrecisionCalculations
Within‐runprecisionsarecalculatedforeachconcentrationseparatelyforeachofthefiveruns.Within‐runprecisionmaybecalculatedusingthedatafromeachrun’striplicateanalysesateachconcentrationas:
%stddeviation
100
Thelargestcalculatedwithin‐run%CVforeachconcentrationwillbeusedtoassesswithin‐runprecisionacceptability.
8.2.2.3.3. Between‐RunPrecisionCalculations
Between‐runprecisioniscalculatedforeachconcentrationoverthefiveruns.Thismaybedonebyusingthecombineddatafromallreplicatesofeachconcentrationas:
%
100
8.2.2.3.4. One‐WayAnalysisofVariance(ANOVA)ApproachtoCalculateCombinedWithin‐RunandBetween‐RunPrecision
Bothwithin‐runandbetween‐runprecisionsmaybecalculatedusingtheone‐wayANOVAapproachwiththevariedfactor(runnumber)asthegroupingvariable.TheANOVAcalculationscanbeeasilyperformedusingaspreadsheetorastatisticalsoftwareprogram.
Usingthisapproach,within‐runprecisionsarecalculatedforeachconcentrationas:
%
100
whereMSwgisthemeansquarewithingroupsobtainedfromtheANOVAtable.
Likewise,between‐runprecisionsarecalculatedas:
%
1 ∗
100
whereMSbgisthemeansquarebetweengroupsobtainedfromtheANOVAtableandnisthenumberofobservationsineachgroup(e.g.,n=3ifdoingtriplicateanalyses).AnnexBprovidesanexampleofhowtheANOVAapproachmaybeusedtocalculatewithin‐runandbetween‐runprecision.
8.3. CalibrationModel
Thecalibrationmodelshallbedeterminedforallquantitativemethods.Thisisaccomplishedbyfirstdeterminingtherangeofanalyteconcentrationsoverwhichthemethodshallbeused,
ASBStandard036,1stEd.‐2017
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sometimescalledtheworkingrange.Withinthisrange,therewillbeacorrelationbetweensignalresponse(e.g.,peakarearatioofanalyteandinternalstandard)andanalyteconcentrationinthesample.Thecalibrationmodelisthemathematicalmodelthatdescribesthiscorrelation.Thechoiceofanappropriatemodel(i.e.,linearorquadratic)isnecessaryforaccurateandreliablequantitativeresults.
Calibratorsamplesareanalyzedtoestablishthecalibrationmodel.Theuseofmatrix‐matchedcalibratorsamplesisencouraged,butnotrequired.Regardlessofthematrixusedtopreparecalibratorsamples,alaboratoryshalldemonstrateacceptablebiasandprecisionwithcontrolsamplespreparedinallmatricesintendedtobeanalyzedbythemethod(seeSection7.2).Forexample,bloodalcoholmethodsmaydemonstrateacceptablebiasandprecisioninwholebloodcontrolsusingaqueouscalibratorsamples.Likewise,acceptablebiasandprecisionmaybedemonstratedusingcalibratorsamplespreparedinwholebloodbutusedtoquantitateanalytesindifferentmatrices(e.g.,postmortemtissues,serum,urine).
Thecalibratorsamplesshallspantherangeofconcentrationsexpected.Atleastsixdifferentnon‐zeroconcentrationsshallbeusedtoestablishthecalibrationmodel.Theconcentrationsshallbeappropriatelyspacedacrossthecalibrationrangetoestablishthemostappropriatecalibrationmodel.Aminimumoffivereplicatesperconcentrationisrequired.Thereplicatestoestablishthecalibrationmodelshallbeinseparateruns.Alldatapointsfromthefiverunsshallbeplottedtogether(usingastatisticalsoftwarepackage)toestablishthecalibrationmodel.Theoriginshallnotbeincludedasacalibrationpoint.
Themostoftenusedcalibrationmodelisthesimplelinearregressionmodelusingtheleastsquaresmethod.However,thismodelisonlyapplicablewhenthereisconstantvarianceovertheentireconcentrationrange.Whenthereisanotabledifferencebetweenvariancesatthelowestandhighestconcentrations,aweightedleastsquaresmodelorotherappropriatenon‐linearmodelshallbeapplied.4Thisisgenerallythecasewhentheconcentrationrangeexceedsoneorderofmagnitude.Ultimately,thebestapproachistousethesimplestcalibrationmodelthatbestfitstheconcentration‐responserelationship.
Althoughithasbecomewidespreadpractice,itisemphasizedthatacalibrationmodelcannotbeevaluatedsimplyviaitscorrelationcoefficient(r).Instead,acalibrationmodelshallbevisuallyevaluatedusingstandardizedresidualplots.Theseallowonetocheckforoutliersthatshallbeeliminatediffoundtobestatisticallysignificant(e.g.,outside±3standarddeviations).Further,residualplotsallowonetodetermineifthevariancesappeartobeequalacrossthecalibrationrangewithasimilardegreeofscatterateachconcentration.Theyalsogiveanindicationifthechosenmodeladequatelyfitsthedata.Forexample,randomdistributionofindividualresidualsaroundthezeroline(homoscedasticity)suggeststhatalinearmodelisappropriate.
Finally,thereareotherappropriatealternativestoevaluatecalibrationmodels(i.e.,ANOVAlack‐of‐fittestforunweightedlinearmodels,checkingforsignificanceofthesecondorderterminquadraticmodels,assessmentofcoefficientofdeterminationforlinearmodels).
Ifalinearcalibrationmodelhasbeenestablished,fewercalibrationsamples(i.e.,fewerlevelsorsingle/fewerreplicates)maybeusedforroutineanalysis.However,iffewercalibrationsamplesarechosen,thesamecalibrators(e.g.,number,replicates,andconcentrationlevel)shallbeusedtoconstructthecalibrationcurvesusedforthebiasandprecisionstudies.Furtherthecalibrationdata
4Ingeneral,non‐linearmodelsmayrequireadditionalcalibratorstoaccuratelycharacterizethecurve.
ASBStandard036,1stEd.‐2017
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shallincludethelowestandhighestcalibrationlevelsusedtoestablishthemodel,aswellasincludenofewerthanfournon‐zerocalibrationpoints.
Additionally,oncethecalibrationmodelisestablishedforavalidatedmethod,itshallnotbearbitrarilychangedtoachieveacceptableresultsduringagivenanalyticalrun.Forexample,oneshallnotswitchfromanunweightedlinearmodeltoaweightedlinearmodelinordertoadjustforchangesininstrumentperformance.
8.4. Carryover
Analytecarryoverintoasubsequentsamplemayleadtoaninaccuratequalitativeorquantitativeresultwhenusinginstrumentalmethods.Carryovershallbeevaluatedduringmethodvalidationintendedforconfirmationand/orquantitation,unlessalaboratoryisconstantlyaddressingcarryoverintheirqualityassurance(QA)/qualitycontrol(QC)practices.
Toevaluatecarryoveraspartofmethodvalidation,blankmatrixsamplesareanalyzedimmediatelyafterahighconcentrationsampleorreferencematerial.Thehighestfortifiedconcentrationatwhichnoanalytecarryoverisobserved(abovethemethod'sLOD)intheblankmatrixsampleisdeterminedtobetheconcentrationatwhichthemethodisfreefromcarryover.Thiscarryoverconcentrationforeachanalyteinthemethodshallbeconfirmedusingtriplicateanalyses.Itisacceptabletolimitthecarryoverstudytothehighestpointofyourcalibrationcurveinquantitativeassays.
Ifpossible,theanalyticalprocedurewillbemodifiedtoremoveanycarryover.Incaseswhenitisnotpossibletoeliminatethecarryover,theSOPshalladdresshowcarryoverwillbemanaged.
8.5. InterferenceStudies
8.5.1. General
Interferingsubstancesfromcommonsourcesshallbeevaluatedinallscreening(exceptimmunoassays),qualitativeidentification,andquantitativemethods.
8.5.2. EvaluatingMatrixInterferences
Wheneverpossible,blankmatrixsamplesfromaminimumoftendifferentsourceswithouttheadditionofaninternalstandard(whenusedinthemethod)shallbeanalyzedtodemonstratetheabsenceofcommoninterferencesfromthematrix.Whilethisapproachmaydetectthemorecommonmatrixinterferences,itisrecognizedthatlesscommoninterferencesmaynotbedetected.
8.5.3. EvaluatingInterferencesfromStable‐IsotopeInternalStandards
Formethodsemployingstableisotopeinternalstandards,theisotopically‐labeledcompoundsmaycontainthenon‐labeledcompoundasanimpurity.Additionally,themassspectraofthelabeledanalogsmaycontainfragmentionswiththesamemass‐to‐chargeratiosasthesignificantionsofthetargetanalyte.Inbothinstances,analyteidentificationorquantitationcouldbeimpacted.
Stable‐isotopeinternalstandardinterferencesshallbeassessedbyanalyzingablankmatrixsamplefortifiedwiththeinternalstandardandmonitoringthesignaloftheanalyte(s)ofinterest.InterferencesbelowtheLODoftheassaymaybeinsignificantdependingonthelaboratory’smission.
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Likewise,ablankmatrixsamplefortifiedwiththeanalyte(s)attheupperlimitofthecalibrationrangeshallbeanalyzedwithoutinternalstandardtoevaluatewhetherrelevantamountsoftheunlabeledanalyteionsappearasisotopically‐labeledcompoundfragmentswhichcouldimpactquantitation.
8.5.4. EvaluatingInterferencesfromOtherCommonlyEncounteredAnalytes
Forallmethodsotherthanimmunoassays,itisnecessarytoevaluateotheranalyteswhichmaybeexpectedtobepresentincasesamplesfortheirpotentialtointerferewiththemethod’sanalytes.Forexample,amethoddevelopedtoanalyzebloodforcocaineshallevaluatewhetherothercommondrugsofabuse,metabolites,andstructurally‐similarcompoundsinterferewiththeassay.Likewise,aheadspacegaschromatograph‐flameionizationdetection(GC‐FID)methoddevelopedforethanolshallevaluatewhetherothercommonvolatileorganiccompoundsinterferewiththeassay.
Thisevaluationisaccomplishedbyanalyzingfortifiedmatrixsamples,previouslyanalyzedcasesamples,orneatreferencematerialsofthepotentialinterference(s)athightherapeuticorlethalconcentrations,dependingontheanalyte,thematrix,andthelaboratory’smission.Themostcommondrugs/metabolitesencounteredinthelaboratoryshallbeincludedintheevaluationtogetherwithothercommondrugswithintheclassification,whereappropriate.
8.6. IonizationSuppression/Enhancement
8.6.1. General
Theenhancementorsuppressionofanalyteionizationresultingfromthepresenceofco‐elutingcompoundsisaphenomenoncommonlyencounteredinliquidchromatography/massspectrometry(LC‐MS)applications.
Whenaveragesuppressionorenhancementoftheanalyte’stargetionoriontransitionandqualifyingions,ifapplicable,exceeds±25%orthe%CVofthesuppressionorenhancementexceeds20%,alaboratoryshalldemonstratethatthereisnoimpactonothercriticalvalidationparameters.Forexample,suppressionorenhancementofionizationismostlikelytoimpactthelimitofdetectionofaqualitativemethod.Likewise,thelimitofdetection,thelimitofquantitation,andbiasmaybeaffectedbyionizationsuppressionorenhancementinquantitativemethods.Theinfluenceontheaboveparametersshallbeassessedbyincreasingthenumberofdifferentsourcesofblankmatricesusedintheirevaluation.Forexample,iftheaveragesuppressionorenhancementexceeds±25%,theLODdeterminationshallbeperformedinmorethanthreeblankmatrices.
Laboratoriesshallalsoassesstheimpactofionizationsuppressionorenhancementonthemethod’sinternalstandards.
Ionizationsuppression/enhancementshallbeevaluatedusingeitheroftheapproachesthatfollow.
8.6.2. Post‐columnInfusiontoAssessIonizationSuppression/Enhancement
Thisapproachprovidesinformationonretentiontimeswhereionizationsuppression/enhancementoccurs.Itisusefulformethoddevelopment,aswellastoassesstheamountofionizationsuppressionorenhancementforLC‐MSbasedconfirmationmethods.Solutionsatbothlowandhighconcentrationsoftheanalyteareindividuallyinfusedwithasyringepumpintotheeluentfromthecolumnviaapost‐column“T”‐connectorandaconstantbaseline
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signalfortheanalyteofinterestismonitored.Wheneverpossible,aminimumoftendifferentprocessedblankmatrixsamplesthatarerepresentativeofthequalityofsamplestypicallyencounteredincaseworkareinjectedintotheLC‐MSduringinfusionofthesolutions.5Ifthereisanyconsiderablesuppressionorenhancement(>25%)oftheinfusedanalytesignalattheretentiontimeoftheanalyte,thenmodificationofthechromatographicsystemorthesamplepreparationmayberequiredtominimizetheeffectofionizationsuppressionorenhancement.
8.6.3. Post‐ExtractionAdditionApproachtoAssessIonizationSuppression/Enhancement
Thisapproachyieldsaquantitativeestimationofionizationsuppression/enhancement.ItisusefulforassessingtheamountofionizationsuppressionorenhancementforLC‐MSbasedquantitativemethods.Twodifferentsetsofsamplesarepreparedandtheanalytepeakareasofneatstandardsarecomparedtomatrixsamplesfortifiedwithneatstandardsafterextractionorprocessing.
Setoneconsistsofneatstandardspreparedattwoconcentrations–onelowandonehigh.Eachoftheseneatstandardsisinjectedaminimumofsixtimestoestablishameanpeakareaforeachconcentration.
Settwoconsistsofaminimumoftendifferentmatrixsources,wheneverpossible.6Eachmatrixsourceisextractedinduplicate.Aftertheextractioniscomplete,eachmatrixsampleisthenreconstituted/fortifiedwitheithertheloworhighconcentrationneatstandard.
Theaverageareaofeachset(X )isusedtoestimatethesuppression/enhancementeffectateachconcentrationasfollows:
%X 2
X 11 100
Twoionizationsuppressionorenhancementpercentageswillbeestablished–oneatthelowconcentrationandoneatthehighconcentration.
8.7. LimitofDetection
8.7.1. General
Limitofdetection(LOD)studiesshallbecarriedoutforallmethods.ThereareanumberofdifferentapproachesfordeterminingtheLOD.Selecttheapproachthatprovidesthemostreasonableestimationofthedetectionlimitgiventheanalyticalinstrumentation(orlackthereof)utilizedinthemethod.
Amethod'sLODincorporatesinstrumentalperformance,aswellasthesamplematrixandinherentprocedurallimitations.Therefore,theLODshallbeassessedovermultiplerunsusingfortifiedmatrixsamplesfromatleastthree(3)differentsourcesofblankmatrix,unlessotherwiseindicatedbelow.Further,whenpossible,itisnecessarytoensurethedefinedLODstillsatisfiesthenecessary
5Additionalmatrixsamplesmayberequiredinpostmortemtoxicologygiventhevarietyofsampleconditionstypicallyencounteredinthiswork.6Additionalmatrixsamplesmayberequiredinpostmortemtoxicologygiventhevarietyofsampleconditionstypicallyencounteredinthiswork.
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parametersforidentification.Forexample,matchingofamassspectrumtoareferencespectrumwithinanacceptablematchfactorcanonlybeachievedbyexperimentaldeterminationofLODratherthantheoreticalcalculation.
TheLODshallbedeterminedbyoneofthefollowingapproaches.
8.7.2. EstimatingLODforaNon‐InstrumentalMethod
Thisapproachismostoftenusedwhenscreeningforthepresenceorabsenceofaspecifiedanalyteorclassofanalytes(e.g.,colortests).ToestimatetheLODforavisual,non‐instrumentalmethod,samplesfortifiedwithdecreasingconcentrationsofanalyteareanalyzedoveraminimumofthreeruns.Whenpossible,multipleanalystsshouldbeinvolvedinestimatingtheLODusingthisapproach.ThelowestconcentrationofanalytethatyieldsapositiveresultonallrunsisconsideredtheLOD.
8.7.3. UsingtheLowestNon‐ZeroCalibratorastheLOD
Thistechniqueisusefulforquantitativemethods.Insomeinstances,itmaybesufficienttodefinetheLODasthevalueofthelowestnon‐zerocalibrator.Aminimumofthreesamplesperrunofthelowestcalibratorshalleachbeanalyzedoverthreerunstodemonstratethatalldetectionandidentificationcriteriaaremet.Ifdesired,itisacceptabletousethesamecalibratorreplicatesusedtoestablishthecalibrationmodel(Section8.3)forsomeofthesamplesusedforthismethod,butadditionalsamples/replicateswillbeneededtomeettheminimumofninedatapoints.
8.7.4. UsingtheDecisionPointConcentrationastheLOD
Thistechniqueisusefulforqualitativeandquantitativemethods.Insomeinstances,itmaybesufficienttodefinetheLODasthevalueofanadministratively‐defineddecisionpoint.Forexample,alaboratorymaychoosetodefineamethod’sLODforethanolas0.02g/dLforbloodbasedonthelaboratory’sadministrativelydefineddecisionpointforreportingthisanalyte,eventhoughalowerLODisanalyticallyachievable.Likewiseforanimmunoassay,alaboratorymaychoosetousethedecisionpointconcentration[thathasdemonstratedappropriateprecision(Section8.2.2.2)]astheassay’sLOD.Aminimumofthreesamplesperrunofafortifiedmatrixsampleattheconcentrationofthedecisionpointshallbeanalyzedoverthreerunstodemonstratethatalldetectionandidentificationcriteriaaremet.ThedatageneratedfortheprecisionatthedecisionpointconcentrationinimmunoassayswillsufficeforthisLODrequirement.
8.7.5. EstimatingLODUsingBackgroundNoise
8.7.5.1. General
TheseapproachesfordeterminingLODareonlyusefulforinstrumentalmethodsthatdemonstratebackgroundnoise.Aminimumofthreedifferentblanksourcematricesshallbeused.Forexample,iftheassayistobeusedforpostmortembloodsamples,threeindependentrepresentativepostmortembloodsourcesareneeded.
8.7.5.2. EstimatingLODUsingReferenceMaterials
Three(ormore)sourcesofblankmatrixsamplesfortifiedatdecreasingconcentrationsareanalyzedinduplicate(twoseparatesamples)foratleastthreeruns.TheLODisconsideredthelowestconcentrationthat1)yieldsareproducibleinstrumentresponsegreaterthanorequalto3.3
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times7thenoiselevelofthebackgroundsignalfromthenegativesamples,and2)achievesacceptablepredefineddetectionandidentificationcriteria(e.g.,retentiontime,peakshape,massspectralionratios).
Whileitmaybepossibletovisuallyassessthesignaltonoiseratio,suchanapproachissubjective.Thereforecalculatethesignal‐to‐noiseratiooruseinstrumentationsoftwaretodeterminetheratio.Ifmanuallycalculated,thesignalisdefinedastheheightresponseoftheanalytepeakandthenoiseisdefinedastheamplitudebetweenthehighestandlowestpointofthebaselineinanareaaroundtheanalytepeak.Eachreplicateshallbeindependentlyevaluated.
‐ ‐
8.7.5.3. EstimatingLODUsingStatisticalAnalysisofBackground
TodeterminetheLODusingthisapproach,aminimumofthreesourcesofblankmatrixareanalyzedinduplicate(twoseparatesamples)overatleastthreeruns.Theaverageandstandarddeviation(sblank)ofthesignal(e.g.,integratedareaofsignalattheanalyte’sretentiontime)fromallblankmatrixsamplesiscalculated.Likewise,fortifiedmatrixsamplesofdecreasingconcentrationareanalyzedinduplicateoverthecourseofatleastthreeruns.Thelowestconcentrationofafortifiedmatrixsamplethatconsistentlyyieldsasignalgreaterthantheaveragesignaloftheblank
matrixsamples(X blank)plus3.3timesthestandarddeviationisidentifiedastheLOD:
LOD=X blank+3.3sblank
8.7.6. EstimatingLODUsingaLinearCalibrationCurve
Thistechniqueisusefulforanyquantitativemethodthatfollowsalinearcalibrationmodel.Aminimumofthreeindependentcalibrationcurvesareconstructedacrosstheworkingrangeoftheanalyticalmethodoverdifferentruns.TheLODcanbeestimatedfromthestandarddeviationoftheyintercept(sy)andtheaverageslope(Avgm)as:
LOD=(3.3sy)/Avgm
8.8. LowerLimitofQuantitation
8.8.1. General
Lowerlimitofquantitation(LLOQ)studiesshallbecarriedoutforallquantitativemethods.Thereareanumberofdifferentapproachesfordeterminingamethod'sLLOQ.Selecttheapproachthatprovidesthemostreasonableestimationofthequantitationlimitgiventheanalyticalinstrumentationutilizedinthemethod.Amethod'sLLOQincorporatesinstrumentalperformance,aswellasthesamplematrixandinherentprocedurallimitations.TheLLOQshallbeassessedovermultiplerunsusingfortified,blankmatrixsamplesfromatleastthreedifferentsourcesofblankmatrix,unlessotherwiseindicatedbelow.
7Useof3.3intheLODcalculationprovidesafalsepositiveerrorrateof0.0005%.(seeBoyd,RobertK.,CeciliaBasic,andRobertA.Bethem.TraceQuantitativeAnalysisbyMassSpectrometry.Hoboken,N.J.:JohnWiley(2008).
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8.8.2. UsingtheLowestNon‐ZeroCalibratorastheLLOQ
Insomeinstances,itmaybesufficienttodefinetheLLOQasthevalueofthelowestnon‐zerocalibrator.Aminimumofthreesamplesperrunofthelowestcalibratorshallbeanalyzedoverthreerunstodemonstratethatalldetection,identification,bias,andprecisioncriteriaaremet.Ifdesired,itisacceptabletousethesamecalibratorreplicatesusedtoestablishthecalibrationmodel(Section8.3)forsomeofthesamplesusedforthismethod,butadditionalsamples/replicateswillbeneededtomeettheminimumofninedatapoints.
8.8.3. UsingDecisionPointConcentrationastheLLOQ
Insomeinstances,itmaybesufficienttodefinetheLLOQasthevalueofanadministratively‐defineddecisionpoint.Forexample,alaboratorymaychoosetodefineamethod’sLLOQforGHBas5mg/Lforantemortembloodbasedonthelaboratory’sadministrativelydefineddecisionpointforreportingthisanalyte,eventhoughalowerLLOQisanalyticallyachievable.Theconcentrationsusedforthisapproachshallremainwithinthepreviouslyestablishedcalibrationcurve.Aminimumofthreesamplesperrunofafortifiedmatrixsampleattheconcentrationofthedecisionpointshallbeanalyzedoverthreerunstodemonstratethatalldetection,identification,bias,andprecisioncriteriaaremet.
8.8.4. EstimatingLLOQUsingReferenceMaterials
Three(ormore)sourcesofblankmatrixsamplesfortifiedatdecreasingconcentrationsareanalyzedinduplicate(twoseparatesamples)overaminimumofthreeruns.Theconcentrationsusedforthisapproachmaybelowerthanthepreviouslyestablishedcalibrationcurve,ifyoudesiretoreportquantitativevaluesunderthelowestpointofyourcalibrationcurve.Inthiscase,biasandprecisionbelowthelowestcalibratorshallbeestablishedwithqualitycontrolsampleswithineachanalyticalbatch.Thelowestconcentrationthatiscapableofachievingacceptabledetection,identification,bias,andprecisioncriteriainallthreefortifiedsamplesisconsideredtheestimatedLLOQ.
9. AdditionalValidationParameters
9.1. General
Incertaininstances,itisimportanttoevaluateadditionalvalidationparameters,ifapplicable.Theseincludeanalytestabilitywhenthematrixisfrozenandthawed,processedsamplestabilityandtheeffectofsampledilutiononbiasandprecision.Alaboratoryshallincludetheseparametersintheirvalidationplan,anddetermineiftheyareapplicabletotheanalyticalmethodoriftheyarealreadyaddressedthroughothermeans(i.e.,qualityassurancepractices,publishedreferences).Thelaboratoryvalidationplanshallincludedocumentationofthisevaluation.
9.2. DilutionIntegrity
Theeffectofsampledilutionshallbedeterminedduringvalidationofquantitativemethodsifthisisaroutinepracticewithinthelaboratory.Attimes,thismaybeduetolowspecimenvolumerequiringthesampleorassaytobeadjustedappropriately.Inotherinstances,excessivelyhighconcentrationsmaybeencounteredthatareabovetheestablishedcalibrationrange.Tobringtheanalyteconcentrationwithinthevalidatedconcentrationrange,thelaboratoryproceduremayallowforreanalysisafterdilutionofthesample.
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Ifdilutionofasampleisallowedbecauseofhighanalyteconcentrationorlowsamplevolume,thenthelaboratoryshallevaluatetheeffectofdilutiononthemethod'sbiasandprecision.Thisisaccomplishedbyrepeatingbiasandwithin‐runprecisionstudiesatcommondilutionratios(e.g.,1:2,1:10,1:50)utilizedbythelaboratoryanddeterminingifperformancecriteriaarestillmet.
9.3. Stability
9.3.1. General
Analytestabilitymaybeaffectedbyanumberofvariablesincludingstorageconditionsandsampleprocessing.Stabilityexperimentsshallbedesignedandcarriedouttoaddresssituationsnormallyencounteredinlaboratoryoperations,unlessanalytestabilityisalreadyaddressedthroughothermeans(i.e.,qualityassurancepractices,publishedreferences).Allstabilitydeterminationsshallincludeasetofsamplespreparedfromreferencematerials.Thereferencematerialsareusedtopreparefortifiedsamplesoftheanalyte(s)atbothlowandhighconcentrationsineachmatrixthatwillbeanalyzedinthemethod.Itisimportantthatalargeenoughvolumeofeachofthesefortifiedsamplesispreparedinordertocompletethestudiesusedinthesectionsbelow.Thesefortifiedsamplesshallinitiallybeanalyzedintriplicatetoestablishtimezeroresponses.Theaveragetimezeroresponseforeachsetofsamplesiscomparedtotheaveragesignalsfromeachofthefollowingstabilitystudies.Linearregressionoftheaveragesignal(e.g.,peakareas8orratiosofpeakareaofanalytetointernalstandard)versustimewillallowforanassessmentoftrends.Alternatively,concentrationsateachtimeintervalcouldbemonitoredprovidedtheconcentrationdeterminationisbasedonthetimezerocalibrationcurve.
9.3.2. Stability–Freeze/Thaw9
Ifitispartofalaboratory’sstandardpracticetofreezesamplespriortoanalysisandtherearenopublisheddatatorelyupon,analytestabilityshallbedeterminedafterthreefreezeandthawcycles.Theabovefortifiedsamples(Section8.3)arealiquotedintoaminimumofthreeseparatestoragecontainersperconcentrationandthenfrozenattheintendedstoragetemperaturefor24hours.Thisisfollowedbyanunassistedthawatroomtemperature.Whencompletelythawed,thefirstsetofsamplesshallbeanalyzedintriplicate,whiletheothersarerefrozenfor12to24hoursunderthesameconditions.Thefreeze/thawcycleandanalysisshallberepeatedtwomoretimes.Theanalyteshallbeconsideredasstableuntiltheaveragesignal(e.g.,peakareaorratiosofpeakareaofanalytetointernalstandard)comparedtothetimezeroaveragesignalfallsoutsideofthemethod’sacceptablebias.Forexample,ifthemethodbiasis±10%andthetimezeroaveragesignalis100,000,theanalyteisconsideredstableuntiltheaveragesignalfallsoutsideofthe90,000–110,000range.
9.3.3. Stability–ProcessedSample
Circumstancesmayariseinwhichsamplesthathaveundergoneroutinepreparationforinstrumentalanalysiscannotbeimmediatelyanalyzed.Itmaybenecessarytorunthesamplethefollowingdayorlater.Intheseinstances,itisimportanttoevaluatethelengthoftimeaprocessed
8Whenmonitoringpeakareas,theinstrument’sresponseshouldbeconstantoverseveraldaysforreliableinterpretationofthedata.9Itisrecognizedthatfreeze/thawandstoragestabilitystudiesinsolidsamples(e.g.,hair,tissues,foodproducts)maynotbepossiblebyfortificationduetothenatureofthesematrices.Cautionshouldbeemployedininterpretingresultsofsolidsampleswhenstabilityinformationisnotavailable.
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samplecanbemaintainedbeforeitundergoesunacceptablechanges,preventingreliableanalytedetection,identification,orquantitation.
TypicallyprocessedsamplesfortifiedperSection8.4arecombinedperconcentrationandthendividedintodifferentautosamplervials.Asindicatedabove,thefirstvialsofeachconcentrationareimmediatelyanalyzedintriplicatetoestablishthetimezeroresponses.Allremainingvialsarestoredinamannerthattheywouldtypicallybestoredduringroutineanalysis(e.g.,refrigerated,atroomtemperatureonautosampler).Theremainingvialsarethenanalyzedintriplicateatdifferenttimeintervals.Averageresponsesateachtimeintervalarecomparedtothetimezeroresponses.Theanalytewillbeconsideredstableuntiltheaveragesignal(e.g.,peakareaorratiosofpeakareaofanalytetointernalstandard)comparedtothetimezeroaveragesignalfallsoutsideofthemethod’sacceptablebias.Forexample,amethod’sbiaslimitis±15%andthetimezeroaveragesignalis100,000.Processedsamplesindifferentautosamplervialsareanalyzedrepeatedlyupto72hours.Theprocessedsample’sanalyteisconsideredstableuntiltheaveragesignalfallsoutsideofthe85,000–115,000range.
10. RequiredRevalidationofPreviouslyValidatedMethods
Modificationstoavalidatedmethodrequireevaluationtoconfirmthatthechangesdonothaveanadverseeffectonthemethod’sperformance.Thedecisionregardingwhichperformancecharacteristicsrequireadditionalvalidationisbasedonconsiderationofthespecificparameterslikelytobeaffectedbythechange(s).Thesechangesmayinclude,butarenotlimitedto:
a) analyticalconditions,
b) instrumentation,
c) sampleprocessing,
d) datasoftware.
Forexample,changesofextractionsolventorbuffermayaffectlinearity,interferences,LLOQ,precision,andbias.Achangeoftheanalyticalcolumnstationaryphaseorachangeinmobilephasecompositionmayaffectlinearityandinterferences.Further,considerationshouldbegiventoconductingparallelstudieswithknownorproficiencysamplesutilizingbothapreviouslyvalidatedmethodandthemodifiedmethodtoevaluatetheeffectsofthechanges.Thegoalistodemonstratetheimpactthechangeshaveontheperformanceofthepreviouslyvalidatedprocedure.
11. DocumentationRequirementsforMethodValidation
Recordkeepingisanessentialpartoflaboratoryoperatingproceduresandisakeycomponentofmethodvalidation.Thedatageneratedduringmethodvalidationstudiesshallbemaintainedandavailableforaudits,reviews,orinspections.Theserecordsshallbeorganizedforeasyretrievalandreview.
Methodvalidationrecordsshallincludeasummaryofthevalidationstudiesconductedandtheirresults.Theformatofthissummaryreportmaybeabriefbulletedreportortablesummaryformattofacilitateaswiftreviewofvalidationstudies.Thesummaryshallminimallyincludethefollowing:
a) scope;
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b) validationplan;
c) descriptionofalltheparametersevaluated,ifanyoftheparameterswerenotevaluated,thenthereasonshallbestatedorjustified;
d) samplepreparationstepstoincludeconcentrationsandmatrices;
e) rawdataorreferencetowheretherawdataarestored;
f) resultsandcalculations;
g) conclusions;
h) references;
i) documentationofmanagementreviewandapproval.
Itisimportantthatthevalidationrecordscontainspecificdetailsregardingthestudiesconducted,including:
a) individualsinvolvedinthemethodvalidation,
b) specificinstrumentation,
c) dates.
Methodvalidationdocumentationshallalsoincludeacopyofthenewlydevelopedanalyticalmethodorareferencetoitslocation.Further,validationdocumentationshouldberetainedforaminimumof10yearsafterthemethodisretired.
12. EfficiencywithValidation
Keepinmindthatsomevalidationexperimentsmaybeconductedconcurrentlywiththesamefortifiedsamples.AnnexD,E,andFpresentexampleapproachestoassistinstreamliningvalidationexperiments.
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AnnexA(informative)
FoundationalPrinciples
Customersofforensicscienceserviceprovidersrequirethatthemethodsusedtoanalyzeevidenceisfit forpurpose. Further,customersneedtoknowthatthemethod'slimitationsareunderstoodwhen used under normal operating conditions. This is one ofmany steps toward ensuring thatqualityresultsareprovidedinlegalmatters.
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AnnexB(informative)
QuantitationofDrugXinBloodValidationExample
Thefollowingisanexampleofsomeofthevalidationstepsoutlinedinthisdocument.Itisnotintendedtoprovidespecificguidanceforanyparticularmethod.
Inthisexample,assumealaboratoryvalidatedaLC/MS/MSmethodforanewopiate,DrugX,inwholeblood.
CreateValidationPlan(Section6)
Beforestartingthevalidationexperiments,thelaboratorypreparedthevalidationplan.Intheplan,theyspecifiedthatanexistingSPEprocedure,alreadyusedfortheextractionofotheropiates,wouldberelieduponforextractingDrugX(Section5).Further,instrumentconditionswerepreviouslyoptimized(Section5),sothoseconditionswerealsolistedintheplan(notshown).Asthisisaquantitativeprocedure,thevalidationparameterslistedinTableB.1—ValidationParameterstobeAssessedwereassessedagainstthelaboratory’spre‐definedacceptancecriteria.
InterferenceStudies(Section8.5)
Ten(10)independentsourcesofblankwholebloodweresecuredfrompreviouslyanalyzedcasestoevaluatematrixinterferences(Section8.5.2).Theblankmatrixsampleswereextractedwithouttheadditionofinternalstandard(d3‐DrugX)andanalyzedusingthenewlydevelopedmethod.NointerferencesattheretentiontimeforDrugXwerenotedafteranalysisoftheblankwholebloodsamples.
Thelaboratoryrandomlyselectedoneoftheblankmatrixsamples,addedd3‐DrugXtothesample(250ng/mL),extractedthesample,andanalyzedit.ThiswastodemonstratethattheinternalstandardwouldnotinterferewiththesignalforDrugX(Section8.5.3).Likewise,anotherrandomblankmatrixsamplewasfortifiedwithDrugXat2000ng/mLandanalyzedwithoutinternalstandard.Thiswastoevaluatewhethertheunlabeledanalyteionsinterferewiththesignalford3‐DrugX.Theresultsdemonstratednointerferencesbetweentheanalyteandinternalstandard.
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TableB.1—ValidationParameterstobeAssessed
Parameter: AcceptanceCriteria:
Bias Shallnotexceed±20%
CalibrationModel 10–1000ng/mL(linearmodeldesired)
Carryover Carryoverafterhighestcalibratordoesnotexceed10%ofsignaloflowestcalibrator
InterferenceStudies Nointerferingsignalfrommatrix,internalstandard,commondrugsofabuse(includingothercommonopiates/metabolites),OTCdrugs,andprescriptionmedications
IonizationSuppression/Enhancement
<25%suppressionorenhancementand<15%CVduetomatrix(ifnot,evaluateimpactonLOD,LLOQ,andBias)
LimitofDetection Shallbe10ng/mLorlower
LowerLimitofQuantitation Shallbe10ng/mLorlower
Precision %CVshallnotexceed20%
DilutionIntegrity Biasandprecisioncriteriashallbemetwithdilutionofsamples.Dilutionratiosevaluatedwilldependonlinearrangeoffinalcalibrationcurve.
ProcessedSampleStability Evaluatelengthoftimethatanalyteinextractedsamplesstoredatroomtemperatureonautosamplerremainsstable
Lastly,toevaluateinterferencesfromothercommonlyencounteredanalytes(Section8.5.4),thelaboratoryinjectedneatsolutionsdilutedinmobilephasetoaconcentrationof5000ng/mL(orhigher)ofallcommonopiatesandmetabolitesobservedintheircasework,othercommonrecreationaldrugsofabuseandtheirmetabolites,othercommonprescriptionmedicationsandtheirmetabolites,andcommonover‐the‐countermedicationsandtheirmetabolites.TableB.2showshowthelaboratoryefficientlypreparedthesesolutionsintofourinjectionstandards.ThelaboratoryobservednointerferenceforthesignalofDrugXord3‐DrugXfromanyofthesecompounds.
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TableB.2—ExampleDrugs/MetabolitesUsedinInterferenceStudy
InjectionMix IncludedDrugs/Metabolites(5000ng/mLunlessnotedotherwise)
OpiatesandRelated codeine,morphine,heroin,6‐acetylmorphine,hydrocodone,hydromorphone,oxycodone,oxymorphone,levorphanol,meperidine,methadone,tramadol,fentanyl
DrugsofAbuse amphetamine,cocaine,benzoylecgonine,ecgoninemethylester,methamphetamine,PCP,MDA,MDMA,THC,THC‐COOH
PrescriptionDrugs antidepressants(amitriptyline,imipramine,doxepin,amoxapine,trazodone,bupropion,fluoxetine,sertraline,citalopram),benzodiazepines(alprazolam,chlordiazepoxide,clonazepam,clorazepate,diazepam),antiarrhythmics(verapamil,diltiazem,lidocaine),barbituratesat500,000ng/mL(amobarbital,butalbital,pentobarbital,phenobarbital),otherCNSdepressants(zopiclone,buspirone,zolpidem)
OTCDrugs antihistamines(diphenhydramine,doxylamine,chlorpheniramine),analgesicsat500,000ng/mL(acetaminophen,ibuprofen),antitussive(dextromethorphan)
CalibrationModel(Section8.3)andCarryover(Section8.4)
Thelaboratoryindicatedadesireforthemethod’scalibrationmodeltobelinearandincludetherangeof10–1000ng/mL.However,toevaluateifthemethodcouldexceedthisrange,thecalibrationsampleswerepreparedinblankbloodattheconcentrationsof10,20,50,100,250,500,1000,1500,and2000ng/mL.Eachcalibratorwasanalyzedonceperruninfiveseparateruns(TableB.3).Anextractedmatrixblankwasanalyzedaftereachcalibratortoevaluatecarryoverateachconcentration.Thedataofallrunswerecombinedintoasinglecalibrationcurve.ItwasnotedthatcarryoverwasnotpresentforDrugXortheinternalstandardinanyoftheextractedblankmatrixsamplesthatfollowedthecalibratorsintherangeof10–1500ng/mL;however,asmallamountofcarryoverforDrugXwasobservedintwoofthefiveblankmatrixsamplesthatfollowedthe2000ng/mLcalibrator.TheintegratedareasofDrugXinthesetwosampleswerelessthan10%ofthesmallestareaofthelowest(10ng/mL)calibrator,sothecarryoverfromthe2000ng/mLcalibratorwasdeemedacceptable.
Thefirstevaluationofthesedatasuggestedthatlinearitymaybreakoffabove1000ng/mL(TableB.3andFigureB.1).Aresidualplotwasusedtofurtherevaluatethesedata(FigureB.2).
TableB.3—CalibrationCurveData
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FigureB.1—CombinedCalibrationCurveDemonstratingLossofLinearityAbove1000G/Ml
TheresidualplotshowedaninvertedU‐shapeddistributionsuggestinganon‐linearmodelwouldbethebestcalibrationmodelforthesedata(FigureB.2).FigureB.2—StandardResidualPlotofCalibrationCurveDatawithanInvertedU‐Shaped
Distribution
Becausethelaboratory’spreferencewastousealinearcalibrationmodel,theyre‐evaluatedthesedataafterdroppingthe1500and2000ng/mLcalibrators.Doingsoallowedfortheiroriginalvalidationplanrequirements(10ng/mL–1000ng/mL)tostillbemet.Therevisedcalibrationcurveappearedtoprovideabetterfitofthesedatausinganunweightedlinearmodel(FigureB.3).Thiswasconfirmedbytheresidualplotthatshowedarandomdistributionaroundthezerolinesuggestingalinearmodelwasthemostappropriateforthesedata(FigureB.4).
R² = 0.9906
0
1
2
3
4
5
6
7
8
0 500 1000 1500 2000
Ratio
Concentration (ng/mL)
‐3
‐2
‐1
0
1
2
3
0 500 1000 1500 2000 2500
Stan
dard Residual
Concentration (ng/mL)
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FigureB.3—RevisedCalibrationCurve
FigureB.4—ResidualPlotofCalibrationCurvedDatawithaRandomDistribution
Forfuturevalidationexperiments,thelaboratoryusedcalibratorspreparedat10,50,100,250,500,and1000ng/mL.
Sinceaccuratequantitativeresultscannotbeassumedabove1000ng/mL,thelaboratoryknewtheywouldhavetore‐extract(withdilution)anysamplesthatexceed1000ng/mL.Therefore,theyplannedtoevaluatedilutionintegrityinratiosupto1:5whenconductingthebiasandprecisionexperiments.
R² = 0.9989
0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
0 200 400 600 800 1000
Ratio
Concentation (ng/mL)
‐3
‐2
‐1
0
1
2
3
0 200 400 600 800 1000
Stan
dard Residual
Concentration (ng/mL)
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Recallthatnocarryoverwasobservedupto1500ng/mLinthelaboratory’sstudy.However,sincetheworkingcalibrationrangewillendat1000ng/mL,thelaboratoryrecognizedthataccuratequantitativeresultscannotbeachievedabovetheworkingrange.Socarryoverwillneedtobeevaluatedinsamplesthatfollowthosethatexceed1000ng/mL.IftheamountofDrugXinsamplesfollowingthosewithconcentrationsgreaterthan1000ng/mLisabovethemethod’sLOD,thesampleswithpotentialcarryoverwillbere‐extractedandanalyzed.
LimitofDetection(Section8.7)
ToestimatetheLOD,thelaboratorychosetoutilizetheresultsfromtheirpreviouslygeneratedcalibrationcurvedata(Section8.7.6).Boththeslopeandy‐interceptoftheindividualcalibrationcurves(10–1000ng/mL)weredeterminedinordertocalculatetheaverageslopeandstandarddeviationofthey‐intercepts(TableB.4).
TableB.4—SlopeandY‐InterceptDatafromCalibrationCurves
Slope y‐interceptRun1 0.003980 ‐0.00050Run2 0.003828 ‐0.01543Run3 0.004009 ‐0.01247Run4 0.003934 0.00695
Run5 0.003995 ‐0.00318Average 0.003949 0.00125StdDev 0.000073 0.01054
TheLODwascalculatedusingtheformula:3.3×0.01054/0.003949=8.8ng/mL
LowerLimitofQuantitation(Section8.8)
ThelaboratorychosetoanalyzereferencematerialstoestablishtheirLLOQ(Section8.8.4).Threesourcesofwholebloodwereeachfortifiedat20,15,and10ng/mL.Theywereextractedandanalyzedinduplicateagainstafreshlypreparedcalibrationcurveonthreedifferentdays.Thelowestconcentrationthatwascapableofreproduciblyprovidingsymmetricalpeaksandtheminimummassspectralidentificationratios,whilemaintainingabiasof±20%anda%CVof<20%wasthe10ng/mLsample.Thisconcentrationwasdeemedasthemethod’sLLOQandreaffirmedacceptableresultsatthelowestcalibrationpoint.
BiasandPrecision(Section8.2)
Toestablishthemethod’sbiasandprecision,thelaboratorypreparedthreepoolsoffortifiedmatrixsamplesatthefollowingconcentrations:low(30ng/mL);medium(400ng/mL);andhigh(800ng/mL).Eachconcentrationpooloffortifiedsampleswasanalyzedintriplicateonfiveseparatedaysalongwithafreshlypreparedcalibrationcurve(TableB.5).
Thelaboratorycalculatedthebias(Section8.2.1)byfirstdeterminingthemeanforeachconcentration.ThisresultedinthevalueslistedinTableB.6.
Fromthesevalues,thebiaswascalculatedateachconcentration.Forexample,forthelowconcentrationsample,thebiaswasdeterminedas:BiasLow=((28–30)/30)×100)=(‐6.7%)
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Likewise,thebiasforthemediumandhighconcentrationswascalculatedas9.3%and–2.4%,respectively.
TableB.5—QuantitativeResults(ng/mL)ofBiasandPrecisionRuns
Low(30ng/mL)
Run1 Run2 Run3 Run4 Run5
Rep1 32 26 29 26 28Rep2 28 24 31 35 25Rep3 27 28 27 30 29Med
(400ng/mL) Run1 Run2 Run3 Run4 Run5Rep1 412 435 427 455 444Rep2 444 410 419 438 442Rep3 422 450 479 452 423High
(800ng/mL) Run1 Run2 Run3 Run4 Run5Rep1 892 793 761 742 820Rep2 827 741 729 734 749Rep3 850 769 803 720 791
TableB.6—MeanConcentrations(ng/mL)forBiasCalculationsConc(ng/mL) CalculatedMean BiasLow(30) 28 ‐6.7%
Med(400) 437 9.3%
High(800) 781 ‐2.4%
Within‐runandbetween‐runprecisionswerecalculatedusingtheone‐wayANOVAapproach(Section8.2.2.3.4).UsingtheANOVA:SingleFactoranalysisinpopularspreadsheetorstatisticsprograms(seeTableB.7forLowConcentration),thelaboratorywasabletoobtainvaluesforthemeansquarewithingroupsforthelowconcentrationandintroducedintotheappropriateformulaasfollows:
%
100
%√7.93328
100
%2.81728
100
% 10.1%
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TableB.7—ANOVACalculationsfor30ng/mLSample
ANOVA:SingleFactor
SUMMARY
Groups Count Sum Average Variance
Column1 3 87 29 7
Column2 3 78 26 4
Column3 3 87 29 4
Column4 3 91 30.33333 20.33333
Column5 3 82 27.33333 4.333333
ANOVA
SourceofVariation
SS df MS F P‐value Fcrit
BetweenGroups
34 4 8.5 1.071429 0.420175 3.47805
WithinGroups 79.33333 10 7.933333
Total 113.3333 14
Thebetween‐runprecisionforthelowconcentrationwascalculatedusingtheformulaandthemeansquarebetweengroupsfromtheANOVAtable:
%
1 ∗
100
%
8.5 3 1 ∗ 7.9333
28100
%2.84528
100
% 10.2%
Usingthedataforthemediumandhighconcentrations,theANOVA:SingleFactoranalysiswasconductedontheselevels(datanotshown)andappropriatevaluesintroducedintotheformulastoobtainthewithin‐runandbetween‐runprecisions.TableB.8liststhecalculatedresultsforallconcentrations.
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TableB.8—PrecisionResults
Low Medium HighWithin‐Run 10.1%CV 4.5%CV 3.9%CV
Between‐Run 10.2%CV 4.2%CV 2.2%CVIonizationSuppression/Enhancement(Section8.6)
AstheinstrumentalportionofthemethodinvolvesLC/MS/MS,thelaboratorywasrequiredtoconductionizationsuppression/enhancementexperiments.Thepost‐columnextractionapproachwaschosen(Section8.6.3).
Twosetsofsampleswerepreparedfortheexperiment.Setoneconsistedofstandardspreparedinmobilephaseat30and800ng/mL.Theywerenotextracted,butinsteadsimplyinjectedsixtimeseach.
Settwowaspreparedintenblankmatrixsamples.Eachblankmatrixwasfromanindependentsourceofblankwholebloodfrompreviouslyanalyzedcases.Thesewerethesametenblankmatrixsamplesusedintheinterferencestudies.Theblankmatrixsampleswereextractedinduplicateandthenfortifiedto30and800ng/mLwithDrugXand250ng/mLwithd3‐DrugX.Eachconcentrationsetsamplewasinjectedonetimeeach.
AveragepeakareasforboththeDrugXandthed3‐DrugXarefoundinTableB.9.
TableB.9—AveragePeakAreasFromSuppression/EnhancementExperiments
AveragePeakAreas30ng/mL 800ng/mL
DrugX d3‐DrugX DrugX d3‐DrugXSet1 13890 110381 330822 112827Set2 11812 102444 303992 105923
Usingtheabovedatasets,thelaboratorycalculatedthe%ionizationsuppression/enhancementforthetargetiontransitionsateachconcentrationusingtheformula:
%Ionizationsuppression/enhancementDrugX(Low)=((11812/13890)‐1)×100=(‐15.0%)
Thenegativevaluesuggestedsomesuppressionwasoccurring,butitwaslessthan25%.
Similarly,thelaboratorycalculatedthe%suppression/enhancementfor800ng/mLandfortheinternalstandardinbothsets.Theresultssuggestedsuppressionof‐8.1%forDrugXatthe800ng/mLconcentration.Althoughatthesameconcentrationinboththelowandhighsamples,thed3‐DrugXdemonstratedionizationsuppressionsof7.2%and6.1%,respectively.
Thedatawerealsousedtocalculatethe%CVateachconcentration.All%CVswere<14%(datanotshown).
Sincetheaveragesuppressionforallanalytesdidnotexceed±25%andthecalculated%CVvaluewas<15%,thevariationwasconsideredinsignificant.Nofurtherworkwasrequiredforothervalidationparameters.
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DilutionIntegrity(Section9.2)
Whilethelaboratoryindicatedthataminimumworkingrangeforthecalibrationcurvewasbetween10–1000ng/mL,theyanticipatedoccasionalsamplesthatcontainDrugXatconcentrationsabove1000ng/mL.Theirinitialattempttoextendthecalibrationrangeto2000ng/mLwasabandonedwhentheyrealizedthatanon‐linearcalibrationmodelwouldbeneeded.Therefore,theyconducteddilutionintegrityexperimentstodemonstrateacceptablebiasandprecisionresultswhensamplesaredilutedindeionizedwater.Theyevaluatedtwodilutionsratios:1:2and1:5.
Thelaboratorypreparedtwofortifiedmatrixsamplesatconcentrationsof1600ng/mLand3000ng/mL.The1600ng/mLsamplewasdiluted1:2beforeextractionandanalysis.Likewise,the3000ng/mLsamplewasdiluted1:5.Bothdilutionsampleswereanalyzedintriplicateoverfivedifferentruns;eachwithafreshlypreparedcalibrationcurve.Biasandprecisioncalculationswereperformedandresults(TableB.10)demonstratedcomparablevaluescomparedtotheresultsobtainedwithoutdilution.Thisprovidedproofofnodetrimentalimpactwhendilutingthesamplesbeforeextraction.
TableB.10—EffectofDilutiononBiasandPrecision
1600ng/mL(1:2dilution)
3000ng/mL(1:5dilution)
Bias 8.2% 9.9%Within‐RunPrecision 4.0% 2.9%Between‐RunPrecision 4.4% 3.7%ProcessedSampleStability(Section9.3)
Thelaboratoryrecognizedthatsamplesarenotalwaysanalyzedimmediatelyafterextractionduetolargebatchesorunforeseendelays.Forexample,theinstrumentmaylosecommunicationwithitscontroller,inadvertentlyshuttingdownabatchrun.Therefore,toevaluatetheimpactofroomtemperaturestorageofprocessedsamplessittingontheautosamplerbeforeanalysis,thelaboratoryconductedastabilitystudyonextractedsamples.Thiswasachievedbypreparingfortifiedmatrixsamplesattwoconcentrations,30ng/mLand800ng/mL.Twelvealiquotsofeachconcentrationwereextracted.Reconstitutedextractsforeachconcentrationwerecombinedandvortexedtoensureadequatemixing.Theconcentrationpoolwasthendividedinto12autosamplervialsandplacedontheautosampler.Thefirstvialofeachlevelwasinjectedthreetimestorepresentthetimezero(t0)sample.Theremainingvialsforeachconcentrationwereanalyzedintriplicateeverysixhoursupto66hours.Analytesignalsfromthetriplicateanalyseswereaveragedandcomparedtothet0signals(TableB.11).
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TableB.11—AveragePeakAreasforProcessedSampleStabilityStudy
Time(hr)AveragePeakArea
30ng/mL 800ng/mL
DrugX d3‐DrugX DrugX d3‐DrugX0 12490 101832 332554 1004236 12289 100382 331820 10032812 12198 100432 330779 10110118 11732 100733 330246 10098724 10983 100992 329787 10083230 10101 101789 326048 10082136 10328 100904 327238 10023442 10281 100086 326838 10032348 10271 100183 315009 9972754 10612 100309 315772 9942160 10402 100233 316231 9638166 10183 100872 315499 94832
ByplottingtheaveragepeakareasforbothDrugXandtheinternalstandard,thelaboratorycouldevaluatetheprocessedsampleswhiletheywerestoredontheautosampler.Astheirrequiredbiasis±20%,theyconsideredthecompoundsstableuntiltheysawadecrease(orincrease)insignalofmorethan20%fromthet0averagepeakarea.Theplotforthe30ng/mLconcentrationofDrugXisshowntodemonstratethisconcept(FigureB.5).
FigureB.5—ChangeinDrugXPeakAreaOver66Hours
5000
6000
7000
8000
9000
10000
11000
12000
13000
14000
15000
0 10 20 30 40 50 60 70
Peak Area
Hours
Drug X ‐ 30 ng/mL
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ThesedataappeartosuggestDrugXremainedstablewithinthepre‐definedlimitsfortheentire66‐hourperiodofthestudy.However,thetrendlineshowsthat66hoursmaybethemaximumperiodoftimebeforethesamplesmayneedtobere‐extracted.Itwasnotedthatatthe30‐hourmark,stabilityseemedtohavedroppedveryclosetothe“instability”point.Sincethepreviouslydeterminedbiaswasactuallymuchbetterthanthe±20%requiredintheirvalidationplan,thelaboratorymadeadecisiontore‐extractanysamplesthatremainontheautosamplermorethan24hours.
DocumentationofResults(Section11)
AlongwithalloftheotherrequireddocumentationlistedinSection11,thelaboratorycomparedtheresultsfromthevalidationstudiesconductedtotheoriginallydefinedrequirements,asdemonstratedinTableB.12.
TableB.12—SummaryofValidationResults
Parameter: AcceptanceCriteria: Result:
Bias Shallnotexceed±20% ‐6.7to9.3%
CalibrationModel 10–1000ng/mL(linearmodeldesired) 10–1000ng/mL(linearmodel)
Carryover Carryoverafterhighestcalibratordoesnotexceed10%ofsignaloflowestcalibrator.
Nosignificantcarryoverat2000ng/mL.Re‐extractandanalyzesamplescontainingDrugXabovetheLODifthatsamplefollowsonethatexceeds1000ng/mLofDrugX.
InterferenceStudies Nointerferingsignalfrommatrix,internalstandard,commondrugsofabuse(includingothercommonopiates/metabolites),OTCdrugs,andprescriptionmedications.
Noobservedinterferencesfrommatrixorfromcommondrugs/metabolites.
IonizationSuppression/Enhancement
<25%suppressionorenhancementand<15%CVduetomatrix(ifnot,evaluateimpactonLOD,LLOQ,andBias).
‐8.1to‐15.0%;<14%CV
LimitofDetection Shallbe10ng/mLorlower 8.8ng/mL
LowerLimitofQuantitation
Shallbe10ng/mLorlower 10ng/mL
Precision %CVshallnotexceed20% Within‐run(3.9to10.1%)
Between‐run(2.2to10.2%)
DilutionIntegrity Biasandprecisioncriteriashallbemetwithdilutionofsamples.
Using1:2and1:5aqueousdilutions,bias(8.2to9.9%)andprecision(within‐run(2.9to4.0%);between‐run(3.7to4.4%).Comparabletoresultsobtainedwithoutdilution.
ProcessedSampleStability
Evaluatelengthoftimethatanalyteinextractedsamplesstoredatroomtemperatureremainsstable
24hours
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AnnexC(informative)
ImmunoassayScreenofBenzodiazepinesinUrineValidationExample
Thefollowingisanexampleoftheimmunoassayvalidationstepsoutlinedinthisdocument.Itisnotintendedtoprovidespecificguidanceforanyparticularmethod.
Inthisexample,assumealaboratoryvalidatedanimmunoassaykitforitsabilitytoscreenurineforbenzodiazepines.
CreateValidationPlan(Section6)
Beforestartingthevalidationexperiments,thelaboratorypreparedthevalidationplan.Intheplan,theyspecifiedthattheywilluseCompanyABC’sELISAImmunoassayKitforBenzodiazepines(Oxazepam)designedwitha“cutoff”of300ng/mL.Thelaboratoryplannedtoselecttheirowncutoffconcentration(decisionpoint)of50ng/mLforthetargetcompoundofoxazepam.Thesamplepreparationsteps,aswellasinstrumentalsettingswerelistedintheplan.Thevalidationparameterswereassessedagainstthepre‐definedrequirementslistedinTableC.1.
TableC.1—ValidationParameterstobeAssessed
Parameter: DesiredLimit:LimitofDetection Sameasdecisionpoint(50ng/mLforoxazepam,lorazepamandalpha‐
hydroxyalprazolamand25ng/mLforalprazolam)
Precision %CVshallnotexceed20%;grandmeans±2StdDevcannotoverlapPrecisionattheDecisionPoint(Section8.2.2.2)
Theproductbrochurelistedthecross‐reactivitiesforoxazepam,otherbenzodiazepines,andtheirmetabolites.Anabbreviatedlistofthesecross‐reactivitiesisasfollows:oxazepam(100%);nordiazepam(425%);lorazepam(175%);alprazolam(450%);andalpha‐hydroxyalprazolam(340%).
Sincetheassaywastobeusedtodeterminetheuseofthebroadclassofbenzodiazepines,thelaboratorywasrequiredtoverifyprecisionforoxazepamandanyotheranalytesthattheychosetoscreenforusingthisassaywithcross‐reactivitieslessthan100%orwithadecisionconcentrationlessthanthatofoxazepam(50ng/mL).Forexample,thislaboratorydecidedtousetheassaytoscreenforlorazepam(decisionconcentration50ng/mL),alprazolam(decisionconcentration25ng/mL)andalpha‐hydroxyalprazolam(decisionconcentration50ng/mL).Bothoxazepamandalprazolamhadtobeevaluatedforprecisionattheirdecisionconcentration.However,sincelorazepamandalpha‐hydroxyalprazolamhadcross‐reactivitiesgreaterthan100%andthesamedecisionpointasoxazepam(50ng/mL),precisionstudieswerenotrequiredfortheseassays.
Thelaboratorypreparedthreepoolsofoxazepam‐fortifiedmatrixsamplesatthefollowingconcentrations:25ng/mL(50%below);50ng/mL(decisionpoint);and75ng/mL(50%above).Eachofthefortifiedsamplesetsisanalyzedintriplicateonfiveseparatedays.TheresultsareshowninTableC.2.
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TableC.2—Results(B/BoforELISA;AbsorbanceValuesForLiquidReagentAssays)ofPrecisionRunsfortheOxazepamSampleSets.
25ng/mL Run1 Run2 Run3 Run4 Run5Rep1 44.396 39.911 39.171 41.213 41.929Rep2 43.896 40.632 37.556 39.111 41.293Rep3 45.323 44.814 39.789 41.454 44.843
GrandMean 41.689StdDev 2.443%CV 5.9%
50ng/mL Run1 Run2 Run3 Run4 Run5Rep1 32.664 31.943 30.649 29.807 32.686Rep2 29.882 30.862 28.210 31.013 32.284Rep3 27.707 31.078 29.619 28.946 29.303
GrandMean 30.444StdDev 1.557%CV 5.1%
75ng/mL Run1 Run2 Run3 Run4 Run5Rep1 19.256 24.012 20.857 23.329 23.342Rep2 17.009 18.928 19.517 20.227 20.831Rep3 17.794 18.712 17.867 20.159 22.003
GrandMean 20.256StdDev 2.152%CV 10.6%
Theresultforthe50ng/mLdecisionpointconcentrationwhenconsideringthegrandmeanofthemeasurementplusorminustwostandarddeviations(30.444±(2×1.557))wasbetween27.330‐33.558.Thisrangedidnotoverlapwiththerangescalculatedforthe25ng/mLor75ng/mLsamples.
The%CVforeachconcentrationwas5.9%,5.1%,and10.6%,respectively;wellbelowtherequirementtonotexceed20%.
Similarexperimentswereconductedforalprazolamatthe25ng/mLdecisionpoint,aswellasconcentrations‐50%and+50%ofthedecisionpoint(datanotshown).
LimitofDetection(Section8.7.4)
Thelaboratoryusedthedecisionpointconcentrationsastheassayslimitofdetectionforeachofthebenzodiazepinesandmetabolites.
DocumentationofResults(Section11)
AlongwithalloftheotherrequireddocumentationlistedinSection11,thelaboratorycomparedtheresultsfromthevalidationstudiesconductedtotheoriginallydefinedrequirements,asdemonstratedinTableC.3.
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TableC.3—SummaryofValidationResults
Parameter: DesiredLimit: Result:LimitofDetection Sameasdecisionpoint:
Oxazepam(50ng/mL)Lorazepam(50ng/mL)Alprazolam(25ng/mL)Alpha‐hydroxyalprazolam(50ng/mL)
50ng/mL50ng/mL25ng/mL50ng/mL
Precision %CVshallnotexceed20%Grandmeans±2StdDevcannotoverlap
5.1to10.6%25ng/mL:36.803–46.57550ng/mL:27.330–33.55875ng/mL:15.952–24.560
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AnnexD(informative)
ExampleFlowchartofMethodValidationExperiments
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AnnexE(informative)
TableofExampleExperimentsforValidationofQualitativeConfirmation/IdentificationMethods
Interference(Section8.5)
10differentsourcesofeachmatrix,noIS 1blanksample+isotopically‐labeledIS 1fortifiedsamplewithhighanalyteconcentrations,noIS Neat,fortified,orauthenticsamplescontainingpotentiallyinterfering
compounds/metabolitesbutnoanalyte
Carryover(Section8.4)
AddressedinroutineQCpracticesbyanalyzingextractedblankmatrixsamplesbetweencasesamples
LimitofDetection(Section8.7.5.2)
Fortifiedmatrixsamplesfortifiedatincreasinglylowerconcentrationsandanalyzedinduplicateover3days.Lowestconcentrationthatreproduciblyyieldssignalgreaterthanorequalto3.3timesthenoiseofbackgroundsignalistheLOD.
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AnnexF(informative)
TableofExampleExperimentsforValidationofQuantitativeMethods
InterferencesIonization
Suppression/Enhancementa CalibrationModel
10differentsourcesofeachmatrix,withoutIS
1blanksamplewithIS 1fortifiedsamplewithhighanalyteconcentrationsandwithoutIS
Neat,fortified,orauthenticsamplescontainingpotentiallyinterferingcompounds/metabolitesbutnoanalyte
Post‐columninfusion: 10blankextractsfortifiedafterextractionatlowconcentration
10blankextractsfortifiedafterextractionathighconcentration
Analytesolutionsforinfusion(lowandhighconcentrations)eachinjected6times
6concentrationlevels,5replicateseach(maybeaccomplishedwithcalibrationcurvesgeneratedforstudiesbelow)
Mainvalidationphase
Bias&Precision DilutionIntegrity
Run Calibration Low Medium High LODb LLOQbBias&Precision
1 6 3 3 3 3 3 3
2 6 3 3 3 3 3 3
3 6 3 3 3 3 3 3
4 6 3 3 3 ‐ ‐ 3
5 6 3 3 3 ‐ ‐ 3aLC‐MS(/MS)methodsonlybForthisexample,thereferencematerialapproachisusedtoestimatetheLOD(Section8.7.5.2)andLLOQ(Section8.8.4)
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AnnexG(informative)
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